Nucleolar Particles Research Articles

The novel ribosome biogenesis inhibitor usnic acid blocks nucleolar pre-60S maturation

The formation of new ribosomes is tightly coordinated with cell growth and proliferation. In eukaryotes, the correct assembly of all ribosomal proteins and RNAs follows an intricate scheme of maturation and rearrangement steps across three cellular compartments: the nucleolus, nucleoplasm, and cytoplasm. We demonstrate that usnic acid, a lichen secondary metabolite, inhibits the maturation of the large ribosomal subunit in yeast. We combine biochemical characterization of pre-ribosomal particles with a quantitative single-particle cryo-EM approach to monitor changes in nucleolar particle populations upon drug treatment. Usnic acid rapidly blocks the transition from nucleolar state B to C of Nsa1-associated pre-ribosomes, depleting key maturation factors such as Dbp10 and hindering pre-rRNA processing. This primary nucleolar block rapidly rebounds on earlier stages of the pathway which highlights the regulatory linkages between different steps. In summary, we provide an in-depth characterization of the effect of usnic acid on ribosome biogenesis, which may have implications for its reported anti-cancer activities.

Principles of human pre-60S biogenesis.

During the early stages of human large ribosomal subunit (60S) biogenesis, an ensemble of assembly factors establishes and fine-tunes the essential RNA functional centers of pre-60S particles by an unknown mechanism. Here, we report a series of cryo-electron microscopy structures of human nucleolar and nuclear pre-60S assembly intermediates at resolutions of 2.5 to 3.2 angstroms. These structures show how protein interaction hubs tether assembly factor complexes to nucleolar particles and how guanosine triphosphatases and adenosine triphosphatase couple irreversible nucleotide hydrolysis steps to the installation of functional centers. Nuclear stages highlight how a conserved RNA-processing complex, the rixosome, couples large-scale RNA conformational changes with pre-ribosomal RNA processing by the RNA degradation machinery. Our ensemble of human pre-60S particles provides a rich foundation with which to elucidate the molecular principles of ribosome formation.

Chromatin network retards nucleoli coalescence

Nuclear bodies are membraneless condensates that may form via liquid-liquid phase separation. The viscoelastic chromatin network could impact their stability and may hold the key for understanding experimental observations that defy predictions of classical theories. However, quantitative studies on the role of the chromatin network in phase separation have remained challenging. Using a diploid human genome model parameterized with chromosome conformation capture (Hi-C) data, we study the thermodynamics and kinetics of nucleoli formation. Dynamical simulations predict the formation of multiple droplets for nucleolar particles that experience specific interactions with nucleolus-associated domains (NADs). Coarsening dynamics, surface tension, and coalescence kinetics of the simulated droplets are all in quantitative agreement with experimental measurements for nucleoli. Free energy calculations further support that a two-droplet state, often observed for nucleoli in somatic cells, is metastable and separated from the single-droplet state with an entropic barrier. Our study suggests that nucleoli-chromatin interactions facilitate droplets’ nucleation but hinder their coarsening due to the coupled motion between droplets and the chromatin network: as droplets coalesce, the chromatin network becomes increasingly constrained. Therefore, the chromatin network supports a nucleation and arrest mechanism to stabilize the multi-droplet state for nucleoli and possibly for other nuclear bodies.

Accumulation and cellular toxicity of aluminum in seedling of Pinus massoniana.

BackgroundMasson pine (Pinus massoniana) is one of the most important timber species with adaptable, fast growing, versatile advantages in southern China. Despite considerable research efforts, the cellular and molecular mechanisms of A1 toxicity and resistance in P. massoniana are still poorly understood. The effects of Al on uptake and translocation of Al and other minerals, cell division and nucleolus in P. massoniana were investigated.ResultsThe results indicated that Al accumulated mainly in the roots, and small amounts were transported to aboveground organs. In the presence of Al, the contents of Mg and Fe in stems increased and decreased in roots. Accumulation of Mn in the organs was inhibited significantly. Evidence from cellular experiments showed that Al had an inhibitory effect on the root growth at all concentrations (10−5 – 10−2 M) used. Chromosome fragments, chromosome bridges, C-mitosis and chromosome stickiness were induced during mitosis in the root tip cells. Al induced the formation of abnormal microtubule (MT) arrays, consisting of discontinuous wavy MTs or short MT fragments at the cell periphery. MT organization and function of the mitotic spindle and phragmoplast were severely disturbed. The nucleolus did not disaggregate normally and still remained its characteristic structure during metaphase. Nucleolar particles containing argyrophilic proteins were accumulated and leached out from the nucleus to the cytoplasm. Evidence confirmed that these proteins contained nucleophosmin (B23), nucleolin (C23) and fibrillarin. Western immunoblot analysis revealed that the contents of three nucleolar proteins increased significantly.ConclusionBased on the information provided in this article, it is concluded that root tips of plants are the most sensitive organ to environmental stresses and the accumulation of Al ions primarily is in roots of P. massoniana, and small amounts of Al are transported to aboveground. Root apical meristems play a key role in the immediate reaction to stress factors by activating signal cascades to the other plant organs. Al induces a series of the cellular toxic changes concerning with cell division and nucleolus. The data presented above can be also used as valuable and early markers in cellular changes induced by metals for the evaluation of metal contamination.

Three major nucleolar proteins migrate from nucleolus to nucleoplasm and cytoplasm in root tip cells of Vicia faba L. exposed to aluminum.

Results from our previous investigation indicated that Al could affect the nucleolus and induce extrusion of silver-staining nucleolar particles containing argyrophilic proteins from the nucleolus into the cytoplasm in root tip cells of Vicia faba L. So far, the nucleolar proteins involved have not been identified. It is well known that nucleophosmin (B23), nucleolin (C23), and fibrillarin are three major and multifunctional nucleolar proteins. Therefore, effects of Al on B23, C23, and fibrillarin in root tip cells of V. faba exposed to 100 μM Al for 48 h were observed and analyzed using indirect immunofluorescence microscopy and Western blotting. The results from this work demonstrated that after 100 μM of Al treatment for 48 h, B23 and C23 migrated from the nucleolus to the cytoplasm and fibrillarin from the nucleolus to the nucleoplasm. In some cells, fibrillarin was present only in the cytoplasm. Western blotting data revealed higher expression of the three major nucleolar proteins in Al-treated roots compared with the control and that the B23 content increased markedly. These findings confirmed our previous observations.

Research on NPM1 gene mutations in acute myeloid leukemia

Nucleolar phosphoprotein (nucleophosmin 1, NPM1), also known as B23, N038, is located in the nucleolar particles of a multifunctional protein widely expressed in various types of cells. At present, a number of studies found that the NPM1 gene mutation is the most frequent acquired molecular genetic abnormalities in acute myeloid leukemia (AML), especially in normal karyotype AML (nk-AML). NPM1 mutation is a special subgroup in AML, which has relatively unique clinical features, and is the independent prognostic indicators of AML. Research on NPM1 mutation has an important clinical significance in the diagnosis, treatment and prognosis judgment of AML patients. This article reviews the discovery of NPM1 gene mutation in AML in recent years, including structure and physiological functions of NPM1 gene, NPM1 gene mutation in AML, detection methods of NPM1 gene mutation, and so on.

A Chaperone for Ribosome Maturation

The nascent pre-rRNA of eukaryotic ribosomes is fully transcribed and assembled into an 80-90 S nucleolar particle before being cleaved into mature ribosomal RNA. The interdependence of steps in the processing of this precursor RNA indicates that RNA processing, at least in part, acts as a quality control mechanism that helps ensure that only functional RNA is incorporated into mature ribosomes. In search of structural components that underlie this interdependence using the Schizosaccharomyces pombe internal transcribed spacer 1 (ITS) as a ligand for affinity chromatography of ITS1-specific proteins, we have isolated a large spliceosome-like protein complex, a ribosome assembly chaperone (RAC) of 20 or more polypeptides (Lalev, A. I., Abeyrathne, P. D., and Nazar, R. N. (2000) J. Mol. Biol. 302, 65-77). When the ITS2 spacer was used in the present study to isolate ITS2-specific proteins, the same proteins were identified consistent with a complex containing multiple specific binding sites. Subsequent competition binding studies indicated that the protein complex actually contains independent binding sites for all four of the transcribed spacers in the pre-rRNA. Because disruption of protein-binding sites in these spacer RNAs is known to severely affect rRNA processing, taken together these results suggest that the RAC complex is a chaperone for ribosome maturation acting as a "rack" on which critical structure is organized.

Identification of an alpha-helical epitope region on the PM/Scl-100 autoantigen with structural homology to a region on the heterochromatin p25beta autoantigen using immobilized overlapping synthetic peptides.

The polymyositis-scleroderma overlap syndrome (PM/Scl) autoantigen is a nucleolar multiprotein particle, presumably participating in the maturation of 5.8S rRNAs. The major target antigens of this particle are two polypeptides with apparent molecular masses of 100 and 75 kDa. In this study we identified the major linear epitopes along the PM/Scl-100 protein sequence by probing overlapping oligopeptides with anti-PM/Scl autoantisera. A major epitope region was identified between amino acids 231 and 245 of the PM/Scl-100 polypeptide. Mutational analysis of the corresponding peptide LDVPPALADFIHQQR by glycine-walk followed by immunodetection of the resulting peptides indicated that amino acids 234, 237, 240, and 241 of the PM/Scl-100 autoantigen are essential for binding of the corresponding antibodies. These results allow us to propose a local alpha-helical secondary structure for the PM/Scl-100 major epitope region. A homology search with the peptide LDVPPALADFIHQQR against the Swiss-Model three-dimensional database reveals some topological homology of the PM/Scl-100 major epitope region with the heterochromatin modifier protein p25beta, a known autoantigen recognized by antibodies from a subset of scleroderma patients.

The planes of division in twin cell doublets

The fifth stage larval epidermis of Calpodes ethlius (Lepidoptera, Hesperiidae) is a syncytium of doublets where sibling cells remain connected by residual midbodies between mitoses. These twins resemble one another more than their other neighbours in features such as the shape and number of nucleolar particles, the number of actin bundles, the position of condensed chromosomes in female cells and the timing of mitosis. Although the patterns of arrangement of structures may be similar in twinned nuclei they differ in their orientation. We have now found that twins also differ in the orientation of their division planes with respect to the previous plane of division. Similarity of structural pattern but not orientation is most easily explained if nuclei, together with determinants for the plane of division, are free to rotate in the plane of the epithelium.

The timing of division in twin cell doublets

The fifth stage larval epidermis of Calpodes ethlius (Lepidoptera. Hesperiidae) is a syncytium of doublets where sibling cells are twins connected by residual midbodies between divisions. Twin cells resemble one another more than their other neighbours in such features as the shape and number of nucleolar particles, the number of actin bundles and the position of condensed female chromosomes. We have now found that they also resemble one another in the timing of cell division in preparation for pupation. Twins are more likely to divide together than at random. The paired timing of mitosis is presumed to be a result of the twins sharing a common cytoplasm.

The relative positions of condensed chromosomes are maintained between divisions in the epidermis of Calpodes ethlius

ABSTRACT The larval epidermis of Calpodes ethlius (Lepidoptera, Hesperiidae) is a syncytium of doublets where sibling cells are twins that remain connected by residual midbodies between mitoses. Twins resemble one another more than their other neighbours in such structural features as the shape and number of nucleolar particles and the number of actin bundles. We have now found that they also resemble one another in the position of the condensed chromosomes that occur in female cells. Female lepidopteran cells contain one or more particles of condensed chromatin, depending on their ploidy. In the epidermis, nuclei with two condensed chromosomes are found in pairs and are separated by the same distances. However, clones of cells with multiple condensed chromosomes are not all alike, suggesting that chromosomes are repositioned at mitosis. Separation distances between chromosomes remain the same between but not through cell divisions, suggesting that determinants for nuclear structure are conserved through interphase and relaxed at mitosis. Although the condensed chromosomes of sibling nuclei resemble one another in their separation, they differ in their orientation, as would be expected if whole nuclei rotate in the plane of the epithelium.

Nucleolar Structural Changes During the Excystment of Onychodromus acuminatus (Ciliophora, Hypotrichia)

Summary The ordered changes which occur in the ultrastructural organization of the nucleoli during excystment of Onychodromus acuminatus have been studied by electron microscopy. The observations were made during the two first hours after the induction of the excystment. During this time many nucleoli lose their granular component and finally disappear. The morphological facts are consistent with the idea that the macronucleus of this ciliate is a very active nucleus during early excystment. It is also suggested that the close relationship which exists between nucleoli and microtubules may represent a functional relationship, facilitating the flow of nucleolar particles from the nucleoli into the cytoplasm where the positioning fibers are forming at this moment and the cortical morphogenesis is being carried out.

Isolation of an MSP1-derived transcriptionally active nucleolar particle

A discrete macromolecular structure with inherent transcription activity has been isolated from Novikoff nucleoli. The particle (350 Å diameter) was released from the intact nucleoli by digestion with restriction endonuclease (Msp1) and is enriched in repetitive DNA sequences both 5′ and 3′ to the ribosomal genes. In vitro transcription analyses suggest that the particle contains the rDNA polymerase template, and that its 12–15 major proteins are responsible for transcriptional activity without added soluble protein factors.

Ultrastructural and purification studies on human tumor nucleolar antigens and nucleolar particles.

The presence of common nucleolar antigens in a broad array of human malignant tumors has led to several lines of investigations. In addition to studies on an increasing number of benign and malignant neoplasms with a variety of antibodies designed to statistically evaluate the presence of nucleolar antigens, purification procedures and chemical analyses are being used to characterize the specific antigens. The localization of the nucleolar antigens in HeLa cells was studied by the postembedding immunoelectron microscopic procedure employing rabbit antibodies to nucleoli or nuclear Tris extracts of these cells. The products of the peroxidase-antiperoxidase complexes visualized by the reaction with diaminobenzidine in nucleoli were mainly found in the nucleolonemas which contain the dense nucleolar RNP components. When these nucleoli became compact after treatment of HeLa cells with adriamycin, the distribution of the immunoreactive products was altered along with distribution of the dense nucleolar components. The human nucleolar antigens were mainly localized to nucleolar regions containing the nucleolar RNP components. Improved purification of the antigens made it possible to provide a satisfactory amino acid analysis of one pI 6.3 antigen. Interestingly, some of the nucleolar antigen was found in miniparticle undescribed until now.

Characterization of proteins from nucleolar preribosomes of mouse leukemia cells by two-dimensional polyacrylamide gel electrophoresis.

Proteins of isolated 80-S and 60-S nucleolar preribosomal particles were characterized by means of two-dimensional polyacrylamide gel electrophoresis, in the lymphocytic mouse leukemia cells L5178Y. Their identification and metabolic relationships with ribosomal subunit proteins were investigated using co-electrophoresis of unlabeled polysomal proteins with labeled proteins of either nucleolar preribosomes or ribosomal subunits. The large and small ribosomal subunits contain 40 and 31 proteins, respectively. The nucleolar 80-S preribosomes were analysed after 2 and 5 h of incubation with tritiated valine and leucine and were shown to contain about 55 proteins. Most of them were identical to cytoplasmic ribosomal subunit proteins. The nucleolar 60-S preribosomes contain all the proteins which are common to 80-S preribosomes and large ribosomal subunits, and one additional protein (L10). The ribosomal proteins which were absent from nucleolar particles were found to be labeled in the cytoplasmic ribosomes after the same incubation period. Thus, in addition to the association of the bulk of ribosomal proteins with 45-S RNA within the 80-S preribosomes, results indicate that a group of ribosomal proteins and particularly from the small subunits, become associated at later stages of the maturation process of mammalian ribosomes. It was further shown that a set of 10 proteins, different from ribosomal polypeptides, were present in nucleolar preribosomal particles. Several of them were associated with polyribosomes in the cytoplasm, whereas the others were unique to the nucleolus.

Characterization by selective labelling and electrophoresis of non-ribosomal polysomal proteins from C11D LM (TK-) cells maintained in exponential growth for 28 h in the presence of actinomycin D (author's transl)

Characterization by Selective Labelling and Electrophoresis of Non-Ribosomal Polysomal Proteins from Cl1D LM (TK−) Cells Maintained in Exponential Growth for 28 h in the Presence of Actinomycin D The partial characterization in vivo of all non-ribosomal polysomal proteins of the mouse cell line Cl1D LM (TK−), by specific labelling of these proteins, is described in this study. 1 We show that Cl1D cells are able to continue their exponential growth for at least 28 h, doubling the initial cell population, in a culture medium containing actinomycin D (0.04–0.06 μg/ml), without inflexion of the growth curve. 2 Under these culture conditions, the synthesis of nucleolar ribosomal particles is completely blocked in the growing cells by actinomycin D. No incorporation of radioactive uridine in the rRNA molecules extracted from the purified polysomes has been found after 8 and 28 h of culture with actinomycin D. The only labelled polysomal RNA was 10–30 S polydisperse on sucrose gradients, as polysomal mRNA would appear. The labelled mRNA · protein particles dissociated from polysomes by EDTA, extend as a heterogeneous peak on CsCl gradients, with densities from 1.42 to 1.52 g/ml. There was no accumulation of labelling at the level of the ribosomal peak. Base Composition of polysomal and 18–55 S nuclear RNA has been determined after the cells had been cultured with and without actinomycin D, and labelled with 32P for 16 h. The distribution of the labelled 2′,3′ nucleotides (after alkaline hydrolysis of RNA and Dowex 1X8 chromatography) demonstrated that no ribosomal RNA was synthesized in the growing cells cultured in the actinomycin D-containing medium: G + C content of polysomal RNA was 43.5%, and that of 18–55 S nuclear RNA was 41%; control values (without actinomycin D) were respectively 58.7% and 57.1%. Total RNA cellular content, as estimated by the orcinol method, was 52–53% of the control, after 28 h of exponential growth with actinomycin D. 3 Using these experimental conditions, long period of protein labelling (28 h) has been used, in an attempt to label only non-ribosomal polysomal proteins and characterize them. Purified polysomes of cells cultured with or without actinomycin D, have been dissociated by EDTA, and the location of labelled proteins has been compared on CsCl gradients. The results obtained indicate that actinomycin D reduces approximately 7 times the protein labelling in the ribosomal peaks, while no obvious change is observed in the labelling level in the mRNA · protein density region. The dissociating effect of 0.3 M to 0.8 M KCl on the labelled polysomal proteins from actinomycin D treated cells has been investigated on CsCl gradients to check where labelled free proteins and polypeptide chains are migrating. These experiments show that their density region is below 1.40 g/ml. The molecular weight of the labelled polysomal proteins was investigated by polyacrylamide-dodecylsulfate electrophoresis. Without actinomycin D, the great majority of labelled polysomal proteins is within a molecular-weight range from about 8000 to 50 000, the known molecular-weight range of structural ribosomal proteins. With actinomycin D, there is practically no labelled material in this molecular-weight range. The labelled proteins are found in the high molecular weight region of the gels, demonstrating that the molecular weight of non-ribosomal polysomal proteins (cytoplasmic polysomal proteins and mRNA bound proteins) are nearly all in the range of 50000 to 150000, or more.

Ribosomal precursor particles in ascites tumor cell nucleoli

Ribonucleoprotein particles have been prepared from isolated nucleoli of ascites tumour cells. The release of nucleolar particles is obtained through the action of heparin. When analysed on sucrose gradients, most of the particles are assignable to the region of 80 S. When the same particles are processed in gradients containing EDTA, the maximum of absorbance at 260 nm is uniformly in the 55 S region. Detailed structural analysis of nucleolar particles was carried out under negative or positive staining in the electron microscope and compared with the finished cytoplasmic ribosomes. The 80 S nucleolar particle measures 230 × 180 Å and displays three striking features: 1. 1. They are often grouped by three or four particles linked by a fine strand of 10 Å. 2. 2. Isolated particles under positive staining are associated with a similar fine strand. 3. 3. Under negative contrast, they appear as tightly coiled irregular structures. On the other hand, the 55 S particle is a loose structure composed of subparticular elements: the average size of this particle is 200 × 170 Å. This 55 S particle is believed to be a dissociation of the 80 S by the action of EDTA. These result are discussed in view of the possible regulatory functions of the nucleolus in the biogenesis of ribosomal RNA and ribosomal peptides.

Identification and metabolic relationship between proteins of nucleolar 60-S particles and of ribosomal large subunits of rat liver by means of two-dimensional disc electrophoresis.

Identification and metabolic relationship between the protein moieties of nucleolar 60-S particles and of ribosomal large subunits of rat liver were investigated by means of two-dimensional poly-acrylamide gel electrophoresis. 1 Cytoplasmic ribosomal large subunits showed 36 major protein spots (C1– C36) on the gel. Similar patterns as a whole were obtained in the case of the nucleolar 60-S particles of thio-acetamide-treated rat liver, although four major proteins (N1–N4) and a number of minor proteins were specific for nucleolar particles and some proteins including three major ones (C8, C20 and C31) for ribosomal large subunits. 2 Gel electrophoresis of the mixture of proteins of large subunits labeled in vivo with 3H-labeled amino acid mixture for 4 h and those of nucleolar 60-S particles labeled with 14C-labeled amino acid mixture for 15 min, both of which were prepared from regenerating rat liver, showed 33 major protein spots having both 3H and 14C radioactivities. While C8, C23 and C31 proteins specific for cytoplasmic ribosomal large subunit showed only 3H radioactivity, proteins specific for nucleolar particles had negligible 14C radioactivity. It was further found that at least two of these three proteins (C20 and C31) were rapidly labeled in vivo when compared with other proteins of the large subunits.

Ribosome assembly in Hela cells.

The 80S nucleolar particles have been found to contain peptides characteristic of 30S as well as 50S ribosomal subunits. This lends support to the idea that the maturation of both types of subunits stems from this 80S particle.

Characterization of ribosomal precursor particles from HeLa cell nucleoli

Ribosomal precursor particles from HeLa cell nucleoli were characterized on the basis of their size, buoyant density and protein content. The nucleolar 80 s and 55 s particles contain more protein than the finished cytoplasmic ribosomes. Reconstruction experiments with excess ribosomal RNA or ribosomal subunits show that the proteins on the nucleolar 80 s and 55 s particles do not arise from non-specific adsorption onto the RNA or protein-protein interaction. The “extra proteins” on the nucleolar particles are distinct from the ribosomal proteins on the basis of their size and labeling kinetics. Similar analysis of nucleolar proteins from the non-particulate regions indicates that these ribosomal proteins flow through the nucleolus while the non-ribosomal polypeptides are re-utilized. The latter may have a role in the processing of nucleolar precursors to cytoplasmic ribosomes.